靶向G-四链体的小分子配体在癌症治疗中应用的研究进展

G-四链体是一类由Hoogsteen氢键维持稳定的,富含鸟嘌呤的DNA或RNA二级结构。人类基因组中存在大量潜在的形成G-四链体的序列,所形成的G-四链体结构能够调控基因组的稳定性、DNA复制和基因表达,其中包括很多与癌症相关基因。因此寻找能够诱导DNA的G富集区域形成G-四链体结构的配体,进而筛选潜在抗癌药物的先导化合物,已成为癌症治疗研究的热点之一。本文对近年来发现和设计的以G-四链体为靶点的小分子配体,按照靶向的G-四链体结构类型和配体的分子结构进行分类,综述了这类化合物在癌症治疗方面的研究进展,分析了相关靶向治疗存在的问题,并对未来的研究方向进行了展望。     

以G-四链体为靶点的小分子端粒酶抑制剂研究进展

富含鸟嘌呤碱基的DNA序列能够通过鸟嘌呤环的互联作用形成四链螺旋结构,这种结构被称为G-四链体。G-四链体由于能够抑制端粒酶的活性而成为抗肿瘤药物的新靶点,能促使G-四链体形成或稳定该结构的物质则可能对癌症有潜在的治疗意义。本文对以G-四链体为靶点的小分子端粒酶抑制剂的研究进行了综述。     

Methods for investigating G-quadruplex DNA/ligand interactions

DNA is considered an important target for drug design and development. Until recently, the focus was on double-stranded (duplex) DNA structures. However, it has now been shown that single stranded DNA can fold into hairpin, triplex, i-motif and G-quadruplex structures. The more interesting G-quadruplex DNA structures comprise four strands of stacked guanine (G)-tetrads formed by the coplanar arrangement of four guanines, held together by Hoogsteen bonds. The DNA sequences with potential to form G-quadruplex structures are found at the chromosomal extremities (i.e. the telomeres) and also at the intra-chromosomal region (i.e. oncogenic promoters) in several important oncogenes. The formation of G-quadruplex structures is considered to have important consequences at the cellular level and such structures have been evoked in the control of expression of certain genes involved in carcinogenesis (c-myc, c-kit, K-ras etc.) as well as in the perturbation of telomeric organization. It has been shown that the formation of quadruplexes inhibits the telomere extension by the telomerase enzyme, which is up-regulated in cancer cells. Therefore, G-quadruplex structures are an important target for drug design and development and there is a huge interest in design and development of small molecules (ligands) to target these structures. A large number of so-called G-quadruplex ligands, displaying varying degrees of affinity and more importantly selectivity (i.e. the ability to interact only with quadruplex-DNA and not duplex-DNA), have been reported. Access to efficient and robust in vitro assays is needed to effectively monitor and quantify the G-quadruplex DNA/ligand interactions. This tutorial review provides an overview of G-quadruplex ligands and biophysical techniques available to monitor such interactions.     

New insights from molecular dynamic simulation studies of the multiple binding modes of a ligand with G-quadruplex DNA

G-quadruplexes are higher-order DNA and RNA structures formed from guanine-rich sequences. These structures have recently emerged as a new class of potential molecular targets for anticancer drugs. An understanding of the three-dimensional interactions between small molecular ligands and their G-quadruplex targets in solution is crucial for rational drug design and the effective optimization of G-quadruplex ligands. Thus far, rational ligand design has been focused mainly on the G-quartet platform. It should be noted that small molecules can also bind to loop nucleotides, as observed in crystallography studies. Hence, it would be interesting to elucidate the mechanism underlying how ligands in distinct binding modes influence the flexibility of G-quadruplex. In the present study, based on a crystal structure analysis, the models of a tetra-substituted naphthalene diimide ligand bound to a telomeric G-quadruplex with different modes were built and simulated with a molecular dynamics simulation method. Based on a series of computational analyses, the structures, dynamics, and interactions of ligand-quadruplex complexes were studied. Our results suggest that the binding of the ligand to the loop is viable in aqueous solutions but dependent on the particular arrangement of the loop. The binding of the ligand to the loop enhances the flexibility of the G-quadruplex, while the binding of the ligand simultaneously to both the quartet and the loop diminishes its flexibility. These results add to our understanding of the effect of a ligand with different binding modes on G-quadruplex flexibility. Such an understanding will aid in the rational design of more selective and effective G-quadruplex binding ligands.     

Structure of human telomeric DNA in crowded solution

G-quadruplex structures formed by DNA at the human telomeres are attractive anticancer targets. Human telomeric sequences can adopt a diverse range of intramolecular G-quadruplex conformations: a parallel-stranded conformation was observed in the crystalline state, while at least four other forms were seen in K(+) solution, raising the question of which conformation is favored in crowded cellular environment. Here, we report the first NMR structure of a human telomeric G-quadruplex in crowded solution. We show that four different G-quadruplex conformations are converted to a propeller-type parallel-stranded G-quadruplex in K(+)-containing crowded solution due to water depletion. This study also reveals the formation of a new higher-order G-quadruplex structure under molecular crowding conditions. Our molecular dynamics simulations of solvent distribution provide insights at molecular level on the formation of parallel-stranded G-quadruplex in environment depleted of water. These results regarding human telomeric DNA can be extended to oncogenic promoters and other genomic G-rich sequences.     

A conformationally constrained nucleotide analogue controls the folding topology of a DNA g-quadruplex

Guanine-rich DNA and RNA sequences can fold into unique structures known as G-quadruplexes. The structures of G-quadruplexes can be divided into several classes, depending on the parallel or antiparallel nature of the strands and the number of G-rich tracts present in an oligonucleotide. Oligonucleotides with single tracts of guanines form intermolecular parallel tetrameric G-quadruplexes. Oligonucleotides with two tracts of guanosines separated by two or more bases can form both intermolecular antiparallel fold-back dimeric and parallel tetrameric G-quadruplexes, and those with four tracts of guanosines can form both intramolecular parallel and antiparallel structures. Intramolecular G-qaudruplexes can fold into several folding topologies including antiparallel crossover basket, antiparallel chair, and parallel propeller. The ability to control the folding of G-quadruplexes would allow the physical, biochemical, and biological properties of these various folding topologies to be studied. Previously, the known methods to control the folding topology of G-quadruplexes included changing the buffer by varying the mono- and divalent cations that are present, and by changing the DNA sequence. Because the glycosidic bonds in the G-quartets of G-quadruplexes with parallel strands are in the anti conformation, we reasoned that incorporation of nucleoside analogues that prefer the anti conformation of the glycosidic bond into G-rich sequences would increase the preference for parallel G-quadruplex formation. As predicted, by positioning the conformationally constrained nucleotide analogue 2'-O-4'-C-methylene-linked ribonucleotide into specific positions of a DNA G-quadruplex we were able to shift the thermodynamically favored structure of a G-quadruplex from an antiparallel to a parallel structure.     

Unique structural features of interconverting monomeric and dimeric G-quadruplexes adopted by a sequence from the intron of the N-myc gene

A multidimensional heteronuclear NMR study has demonstrated that a guanine-rich DNA oligonucleotide originating from the N-myc gene folds into G-quadruplex structures in the presence of K(+), NH(4)(+), and Na(+) ions. A monomeric G-quadruplex formed in K(+) ion containing solution exhibits three G-quartets and flexible propeller-type loops. The 3D structure with three single nucleotide loops represents a missing element in structures of parallel G-quadruplexes. The structural features together with the high temperature stability are suggestive of the specific biological role of G-quadruplex formation within the intron of the N-myc gene. An increase in K(+) ion and oligonucleotide concentrations resulted in transformation of the monomeric G-quadruplex into a dimeric form. The dimeric G-quadruplex exhibits six stacked G-quartets, parallel strand orientations, and propeller-type loops. A link between the third and the fourth G-quartets consists of two adenine residues that are flipped out to facilitate consecutive stacking of six G-quartets.     

Telomestatin and diseleno sapphyrin bind selectively to two different forms of the human telomeric G-quadruplex structure

The human telomeric sequence d[T(2)AG(3)](4) has been demonstrated to form different types of G-quadruplex structures, depending upon the incubation conditions. For example, in sodium (Na(+)), a basket-type G-quadruplex structure is formed. In this investigation, using circular dichroism (CD), biosensor-surface plasmon resonance (SPR), and a polymerase stop assay, we have examined how the addition of different G-quadruplex-binding ligands affects the conformation of the telomeric G-quadruplex found in solution. The results show that while telomestatin binds preferentially to the basket-type G-quadruplex structure with a 2:1 stoichiometry, 5,10,15,20-[tetra-(N-methyl-3-pyridyl)]-26-28-diselena sapphyrin chloride (Se2SAP) binds to a different form with a 1:1 stoichiometry in potassium (K(+)). CD studies suggest that Se2SAP binds to a hybrid G-quadruplex that has strong parallel and antiparallel characteristics, suggestive of a structure containing both propeller and lateral, or edgewise, loops. Telomestatin is unique in that it can induce the formation of the basket-type G-quadruplex from a random coil human telomeric oligonucleotide, even in the absence of added monovalent cations such as K(+) or Na(+). In contrast, in the presence of K(+), Se2SAP was found to convert the preformed basket G-quadruplex to the hybrid structure. The significance of these results is that the presence of different ligands can determine the type of telomeric G-quadruplex structures formed in solution. Thus, the biochemical and biological consequences of binding of ligands to G-quadruplex structures found in telomeres and promoter regions of certain important oncogenes go beyond mere stabilization of these structures.     

Ligand Design to Acquire Specificity to Intended G-Quadruplex Structures

A G-quadruplex is a nucleic acid secondary structure that is adopted by guanine-rich sequences, and is considered to be relevant in various pharmacological and biological contexts. G-Quadruplexes have also attracted great attention in the field of DNA nanotechnology because of their extremely high thermal stability and the availability of many defined structures. To date, a large repertory of DNA/RNA G-quadruplex-interactive ligands has been developed by numerous laboratories. Several relevant reviews have also been published that have helped researchers to grasp the full scope of G-quadruplex research from its outset to the present. This review focuses on the G-quadruplex ligands that allow targeting of specific G-quadruplexes. Moreover, unique ligands, successful methodologies, and future perspectives in relation to specific G-quadruplex recognition are also addressed.     

Two-repeat human telomeric d(TAGGGTTAGGGT) sequence forms interconverting parallel and antiparallel G-quadruplexes in solution: distinct topologies, thermodynamic properties, and folding/unfolding kinetics

We demonstrate by NMR that the two-repeat human telomeric sequence d(TAGGGTTAGGGT) can form both parallel and antiparallel G-quadruplex structures in K(+)-containing solution. Both structures are dimeric G-quadruplexes involving three stacked G-tetrads. The sequence d(TAGGGUTAGGGT), containing a single thymine-to-uracil substitution at position 6, formed a predominantly parallel dimeric G-quadruplex with double-chain-reversal loops; the structure was symmetric, and all guanines were anti. Another modified sequence, d(UAGGGT(Br)UAGGGT), formed a predominantly antiparallel dimeric G-quadruplex with edgewise loops; the structure was asymmetric with six syn guanines and six anti guanines. The two structures can coexist and interconvert in solution. For the latter sequence, the antiparallel form is more favorable at low temperatures (<50 degrees C), while the parallel form is more favorable at higher temperatures; at temperatures lower than 40 degrees C, the antiparallel G-quadruplex folds faster but unfolds slower than the parallel G-quadruplex.     

利用对G-四链体环部的构型调节进行传感器的设计

对鸟嘌呤碱基G重复序列之间连接环结构对G-四链体形成的影响进行了研究。发现在连接环较长,DNA链不易形成G-四链体的情况下,可以通过将环序列设计成双链结构的方式促进G-四链体的重新形成。这就为传感器的设计提供了一个新途径,即可以利用目标分子对环部双链的调节作用控制G-四链体DNA酶的活性。为证明这一点,在双链区域引入T-T碱基错配,破坏双链结构使DNA链不能形成G-四链体。Hg2+对T-T错配的稳定作用可以促进双链结构的形成,DNA链重新折叠成G-四链体,得到的G-四链体与氯化血红素(Hemin)结合后形成具有过氧化物酶活性的G-四链体DNA酶,据此构建了Hg2+传感器。利用此传感器可在10~700 nmol/L范围内实现Hg2+的定量检测,检出限为8.7 nmol/L。在此基础上,利用半胱氨酸可以将Hg2+从T-Hg2+-T碱基对上竞争下来的能力,设计了一种半胱氨酸的检测方法。此方法可以在20~600 nmol/L范围内实现半胱氨酸的定量检测,检出限为14 nmol/L。     

Direct NMR detection of alkali metal ions bound to G-quadruplex DNA

We describe a general multinuclear (1H, 23Na, 87Rb) NMR approach for direct detection of alkali metal ions bound to G-quadruplex DNA. This study is motivated by our recent discovery that alkali metal ions (Na+, K+, Rb+) tightly bound to G-quadruplex DNA are actually "NMR visible" in solution (Wong, A.; Ida, R.; Wu, G. Biochem. Biophys. Res. Commun. 2005, 337, 363). Here solution and solid-state NMR methods are developed for studying ion binding to the classic G-quadruplex structures formed by three DNA oligomers: d(TG4T), d(G4T3G4), and d(G4T4G4). The present study yields the following major findings. (1) Alkali metal ions tightly bound to G-quadruplex DNA can be directly observed by NMR in solution. (2) Competitive ion binding to the G-quadruplex channel site can be directly monitored by simultaneous NMR detection of the two competing ions. (3) Na+ ions are found to locate in the diagonal T4 loop region of the G-quadruplex formed by two strands of d(G4T4G4). This is the first time that direct NMR evidence has been found for alkali metal ion binding to the diagonal T4 loop in solution. We propose that the loop Na+ ion is located above the terminal G-quartet, coordinating to four guanine O6 atoms from the terminal G-quartet and one O2 atom from a loop thymine base and one water molecule. This Na+ ion coordination is supported by quantum chemical calculations on 23Na chemical shifts. Variable-temperature 23Na NMR results have revealed that the channel and loop Na+ ions in d(G4T4G4) exhibit very different ion mobilities. The loop Na+ ions have a residence lifetime of 220 micros at 15 degrees C, whereas the residence lifetime of Na+ ions residing inside the G-quadruplex channel is 2 orders of magnitude longer. (4) We have found direct 23Na NMR evidence that mixed K+ and Na+ ions occupy the d(G4T4G4) G-quadruplex channel when both Na+ and K+ ions are present in solution. (5) The high spectral resolution observed in this study is unprecedented in solution 23Na NMR studies of biological macromolecules. Our results strongly suggest that multinuclear NMR is a viable technique for studying ion binding to G-quadruplex DNA.     

Characterization of structure and stability of long telomeric DNA G-quadruplexes

In the current study, we used a combination of gel electrophoresis, circular dichroism, and UV melting analysis to investigate the structure and stability of G-quadruplexes formed by long telomeric DNAs from Oxytricha and human, where the length of the repeat (n)=4 to 12. We found that the Oxytricha telomeric DNAs, which have the sequence (TTTTGGGG)n, folded into intramolecular and intermolecular G-quadruplexes depending on the ionic conditions, whereas human telomeric DNAs, which have the sequence (TTAGGG)n, formed only intramolecular G-quadruplexes in all the tested conditions. We further estimated the thermodynamic parameters of the intramolecular G-quadruplex. We found that thermodynamic stabilities of G-quadruplex structures of long telomeric DNAs (n=5 to 12) are mostly independent of sequence length, although telomeric DNAs are more stable when n=4 than when n>or=5. Most importantly, when n is a multiple of four, the change in enthalpy and entropy for G-quadruplex formation increased gradually, demonstrating that the individual G-quadruplex units are composed of four repeats and that the individual units do not interact. Therefore, we propose that the G-quadruplexes formed by long telomeric DNAs (n>or=8) are bead-on-a-string structures in which the G-quadruplex units are connected by one TTTT (Oxytricha) or TTA (human) linker. These results should be useful for understanding the structure and function of telomeres and for developing improved therapeutic agents targeting telomeric DNAs.     

Cationic metal-corrole complexes: design, synthesis, and properties of guanine-quadruplex stabilizers

A series of pyridinium and quaternary ammonium copper corroles has been designed and synthesized. All new compounds have been fully characterized by NMR spectroscopy, high-resolution mass spectrometry, UV/Vis spectrscopy, and elemental analysis. Biochemical studies have indicated that all of these corrole derivatives can stabilize G-quadruplex structures, with corrole 4 being the most effective according to the results of circular dichroism (CD) melting experiments, polymerase chain reaction (PCR) stop assays, and surface plasmon resonance (SPR) experiments. Moreover, both corroles 3 and 4 tend to induce the human telomeric sequence to form hybrid G-quadruplex structures, whereas corroles 8 and 9 are more inclined to induce the human telomeric sequence to form antiparallel G-quadruplex structures.     

Synthesis and Structural Characterization of Stable Branched DNA G-Quadruplexes Using the Trebler Phosphoramidite

Guanine (G)-rich sequences can form a noncanonical four-stranded structure known as the G-quadruplex. G-quadruplex structures are interesting because of their potential biological properties and use in nanosciences. Here, we describe a method to prepare highly stable G-quadruplexes by linking four G-rich DNA strands to form a monomolecular G-quadruplex. In this method, one strand is synthesized first, and then a trebler molecule is added to simultaneously assemble the remaining three strands. This approach allows the introduction of specific modifications in only one of the strands. As a proof of concept, we prepared a quadruplex where one of the chains includes a change in polarity. A hybrid quadruplex is observed in ammonium acetate solutions, whereas in the presence of sodium or potassium, a parallel G-quadruplex structure is formed. In addition to the expected monomolecular quadruplexes, we observed the presence of dimeric G-quadruplex structures. We also applied the method to prepare G-quadruplexes containing a single 8-aminoguanine substitution and found that this single base stabilizes the G-quadruplex structure when located at an internal position.     

Platinum(II)-Based Optical Probes for Imaging Quadruplex DNA Structures via Phosphorescence Lifetime Imaging Microscopy

G-quadruplex DNA is a non-canonical structure that forms in guanine-rich regions of the genome. There is increasing evidence showing that G-quadruplexes have important biological functions, and therefore molecular tools to visualise these structures are important. Herein we report on a series of new cyclometallated platinum(II) complexes which, upon binding to G-quadruplex DNA, display an increase in their phosphorescence, acting as switch-on probes. More importantly, upon binding to G-quadruplexes they display a selective and distinct lengthening of their emission lifetime. We show that this effect can be used to selectively visualise these structures in cells using Phosphorescence Lifetime Imaging Microscopy (PLIM).     

Selective G-quadruplex DNA stabilizing agents based on bisquinolinium and bispyridinium derivatives of 1,8-naphthyridine

Various biologically relevant G-quadruplex DNA structures offer a platform for therapeutic intervention for altering the gene expression or by halting the function of proteins associated with telomeres. One of the prominent strategies to explore the therapeutic potential of quadruplex DNA structures is by stabilizing them with small molecule ligands. Here we report the synthesis of bisquinolinium and bispyridinium derivatives of 1,8-naphthyridine and their interaction with human telomeric DNA and promoter G-quadruplex forming DNAs. The interactions of ligands with quadruplex forming DNAs were studied by various biophysical, biochemical, and computational methods. Results indicated that bisquinolinium ligands bind tightly and selectively to quadruplex DNAs at low ligand concentration (～0.2-0.4 μM). Furthermore, thermal melting studies revealed that ligands imparted higher stabilization for quadruplex DNA (an increase in the T(m) of up to 21 °C for human telomeric G-quadruplex DNA and >25 °C for promoter G-quadruplex DNAs) than duplex DNA (ΔT(m) ≤ 1.6 °C). Molecular dynamics simulations revealed that the end-stacking binding mode was favored for ligands with low binding free energy. Taken together, the results indicate that the naphthyridine-based ligands with quinolinium and pyridinium side chains form a promising class of quadruplex DNA stabilizing agents having high selectivity for quadruplex DNA structures over duplex DNA structures.     

Cation-mediated energy transfer in G-quadruplexes revealed by an internal fluorescent probe

A single pyridine unit incorporated into G-quadruplex DNA has revealed efficient energy transfer reactions in cation-containing G-quadruplexes. 8-(2-Pyridyl)-2'-deoxyguanosine, "2PyG", is a highly sensitive internal fluorescent probe of G-quadruplex folding and energy transfer. 2PyG was minimally disruptive to G-quadruplex folding and exhibited intense fluorescence, even when it was base-stacked with other guanine residues. Using 2PyG we have quantified energy transfer efficiencies within G-quadruplex structures prepared under conditions of excess Na(+)/K(+) (110 mM) or in 40% polyethylene glycol (PEG) under salt deficient conditions. G-quadruplex structures containing coordinated cations exhibited efficient DNA-to-probe energy transfer reactions (η(t) = 0.11-0.41), while PEG-folded G-quadruplexes exhibited very little energy transfer (η(t) = 0.02-0.07). Experiments conducted using unmodified G-quadruplexes suggest that cation coordination at the O(6) position of guanine residues results in enhanced quantum yields of G-quadruplex nucleobases that, in turn, serve as efficient energy donors to 2PyG. Given the growing interest in G-quadruplex-based devices and materials, these results will provide important design principles toward harnessing the potentially useful photophysical properties of G-quadruplex wires and other G-rich structures.     

Triggering G-Quadruplex Conformation Switching with [7]Helicenes

The dynamic interplay between two types of chiral structures; fully conjugated racemic hetero[7]helicenes and DNA strands prone to fold into G-quadruplex structures is described. Both the [7]helicenes and the G-quadruplex DNA structures exist in more than one conformation in solution. We show that the structures interact with and stabilise each other, mutually amplifying and stabilising certain conformations at increased temperatures. The [7]helicene ligands L1 and L2 stabilise the parallel conformation of k-ras significantly, whereas hybrid (K⁺) and antiparallel (Na⁺) h-telo G-quadruplexes are stabilised upon conformational switching into altered G-quadruplex conformations. Both L1 and L2 induce parallel G-quadruplexes from hybrid structures (K⁺) and L1 induces hybrid G-quadruplexes from antiparallel conformations (Na⁺). Enantioselective binding of one helicene enantiomer is observed for helicene ligand L2 , and VTCD melting experiments are used to estimate the racemisation barrier of the helicene.